La Jolla, CA – Researchers have long wondered how nuclear pores – the all-important channels that control the flow of information in and out of a cell's nucleus – double in number to prepare for the split to come when a cell divides. Now, for the first time, scientists at the Salk Institute for Biological Studies watched as new funnel-like pore structures formed from scratch, and inserted themselves into the nuclear membrane.

This discovery adds to the picture of how a cell divides in such a way that the genome (genetic blueprint) encased inside the nucleus can continue communicating with the rest of the cell. "This issue is as important to understanding the cell cycle as is the question of how DNA replicates," says Martin Hetzer, Ph.D., an assistant professor in the Molecular and Cell Biology Laboratory and lead author of the study published in the April 21 issue of the journal Science.

Nuclear pores are gigantic structures that control the transport of molecules such as RNA and protein in and out of a cell's inner sanctum, the nucleus, which safeguards the cell's genomic brain. All chemical reactions that occur in a cell emanate from the genes within the nucleus. "Maybe not surprisingly, any disturbance in the flow of information across the nuclear membrane can alter cell functioning," says Hetzer.

"Nuclear pores are truly amazing," says postdoctoral researcher and co-first author Maximiliano D'Angelo, Ph.D. "They are the biggest protein structures within a cell and control the entire traffic in and out of the cell's nucleus, from tiny molecules such as histones, which bind DNA, to huge structures such as ribosomes," he explained.

To form the transport channels that span the nuclear membrane, 30 different proteins, called nucleoporins, come together in an orderly fashion and insert themselves into the nuclear envelope, where they form eight-fold symmetrical nuclear pore complexes. Each protein is present in copies of eight or multiples of eight, forming a structure that contains several hundred proteins and is 30 times the size of a ribosome, the cellular protein factory. "But how nucleoporins find their way into the nuclear membrane and whether existing pores serve as templates had been unknown," says D'Angelo.

To study this process, the Salk researchers created a cell-free system based on frog's eggs (oocytes) that was able to recapitulate the insertion of the nuclear pore complex into the nuclear membrane. Using advanced real-time imaging tools the scientists watched as a nuclear membrane – pores and all – formed within an hour.

"We were able to visualize single nuclear pore complexes," says graduate student and co-first author Daniel Anderson. "This allowed us not only to watch as single pores formed but also to demonstrate that they formed from scratch without the help of already existing pores."

In another experiment, the group used four-dimensional confocal microscopy to follow the formation in cultured human cells of a single pore that had been labeled with a fluorescent tag. If the nuclear pore had split to give rise to two daughter pores, two bright dots would have emerged from one; however, the researchers tracked movement of only one dot, confirming their previous finding that pores formed from scratch.

Additional research demonstrated that nuclear pore assemblies are added in a stepwise, coordinated process requiring components on both sides of the nuclear membrane. "This has important consequences for the next big issue – the question of how these structures all fuse together," Hetzer says.

The Salk Institute for Biological Studies in La Jolla, California, is an independent nonprofit organization dedicated to fundamental discoveries in the life sciences, the improvement of human health and the training of future generations of researchers. Jonas Salk, M.D., whose polio vaccine all but eradicated the crippling disease poliomyelitis in 1955, opened the Institute in 1965 with a gift of land from the City of San Diego and the financial support of the March of Dimes.